Beta amino acid-modified and fluorescently labelled kisspeptin analogues with potent KISS1R activity

Article abstract of DOI:10.1002/psc.2883

Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a du

Full text of DOI:10.1002/psc.2883

Research Article
Received: 9 February 2016
Revised: 11 March 2016
Accepted: 12 March 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2883
Beta amino acid-modified and fluorescently
labelled kisspeptin analogues with potent
KISS1R activity
a
a
b
b
a
M. A. Camerino, M. Liu, S. Moriya, T. Kitahashi, A. Mahgoub,
a
a
b
b
a
S. J. Mountford, D. K. Chalmers, T. Soga, I. S. Parhar and P. E. Thompson *
Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R
system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a
dual luciferase reporter assay in KISS1R-transfected HEK293T cells. In general terms the results reflect the outcomes of other assay
2
formats and a number of potent agonists were identified among the analogues, including β -hTyr-modified and fluorescently la-
belled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity
within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley &
Sons, Ltd.
Keywords: kisspeptin; KISS1R agonist; beta amino acids; fluorescent peptides
pentapeptides (FTM series) from Tomita et al.[12] Just one peptide
antagonist has been reported known as Peptide 234.[15] Most re-
Introduction
Kisspeptin (aka metastin) and its cognate receptor, KISS1R (GPR54),
have been identified in various vertebrate species [1–3] including
humans [4]. Kisspeptin was found to present as several forms
cently, a triazol-linked family of analogues has been described in-
cluding peptide ‘Beltramo 3’ (Table 1).[16]
While giving promise to the idea of the development of thera-
peutic agents targeting GPR54, these few reports leave us with little
information about the interaction between kisspeptin and GPR54.
Structural information of kisspeptins is limited to conformational
studies of kisspeptin-13 that suggests a helical conformation of
the peptide in solution, [9] while in contrast certain modifications
are consistent with favouring turn conformers. [14] In the long term,
the identification of GPR54-dependent biological functions will be
well served by developing a better understanding of the bioactive
conformation and how it interacts with the receptor. This has been
made more significant with the apparent affinity of kisspeptin pep-
tides for the more recently identified neuropeptide FF receptors.[17]
Peptidomimetic kisspeptin analogues (agonists and antagonists)
are essential tools to improve our knowledge in this area.
The in vitro study of GPR54 agonsim has been achieved via a
number of different assay formats. The majority of studies have
employed calcium flux in transfected-CHO cells as measured in
the FLIPR assay protocols. [12] Other assays have examined
ERK1/2 phosphorylation. [13] Reporter genes have also been
employed. First, Niida reported a LacZ-based system in yeast, [18]
while Lee et al. utilised a c-fos-Luciferase system. [1] Kuohung
(KP-10, 13, 15, and KP-54) derived from a precursor peptide, which
share a functionally important N-terminal core region. Kisspeptin’s
activity [5] is dictated through the KISS1 receptor (GPR54) and is a
pivotal element in the neuroendocrine network governing gonad-
otropin secretion and is thus essential for the physiological func-
tions of gonadotropin–releasing hormone (GnRH). Thus KISS1R
agonists may be useful for the treatment of infertility,
hypogonadism, and delayed puberty, and functional antagonists
would be useful for the treatment of hormone – dependent can-
cers (prostate, breast, endometrial), ovarian hyperstimulation, con-
traception and precocious puberty.[6–8] However, despite recent
publications, much remains unknown about the physiological
role(s) and mechanisms of actions of the ‘kisspeptin family of pep-
tides’ and associated receptors in veterinary and aquaculture set-
tings. The dearth of information about how kisspeptin interacts
with GPR54 is due to, in-part, limited access to appropriate
kisspeptin ‘mimics’ for use as biological probes and suitable bio-
logical assays.
Like most proteins and peptides, the utility of kisspeptin and its
peptide analogues either clinically or as pharmacological tools will
be compromised by limiting physicochemical properties. Typically
this means poor oral bio-availability and its short biological half-life,
thus mimics or antagonists necessarily need to avoid these short-
comings. Approaches to novel GPR54 ligands (agonists and antag-
onists) are still in their infancy but include the following: (i) HTS for
small molecules GPR54 agonists and antagonists [9–11] and (ii)
Peptide analogues designed with unnatural amino acid substitu-
tions (Table 1).[12] Among these, a series of agonist compounds
*
Correspondence to: P. E. Thompson, Medicinal Chemistry, Monash Institute of
Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052
Australia. E-mail: philip.thompson@monash.edu
a Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash
University, 381 Royal Parade, Parkville 3052, Australia
1
have been described including d-Tyr -KP10,[13] other analogues
b Brain Research Institutes, Monash University Malaysia, Jalan Lagoon Selatan,
by workers at Takeda (TAK series)[14] and N-terminally modified
Bandar Sunway, Selangor, 47500, Malaysia
J. Pept. Sci. 2016; 22: 406–414
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

KISSPEPTIN ANALOGUES
Table 1. Reported KISS1R ligands
Name
Sequence
Reference
Kisspeptin-10
H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
H-d-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
d-Tyr-d-Pya(4)-Asn-Ser-Phe-azaGly-Leu-Arg(Me)-Phe-NH
Ac-d-Tyr-Hyp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH
Ac-d-Tyr-d-Trp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH
FBz-Phe-Gly-Leu-Arg-Phe-NH
FBz-Phe-Gly = Leu-Arg-Phe-NH
Ac-Tyr-Asn-Trp-Asn-Ser-Phe-Glyψ[Tz]Leu-Arg-Phe-NH
Ac-d-Ala-Asn-Trp-Asn-Gly-Phe-Gly-d-Trp-Arg-Phe-NH
2
[dY]1-KP10
2
[13]
[14]
[14]
[14]
[12]
[12]
[16]
[15]
a
KISS1-305
TAK448
2
2
TAK663
2
FTM-080
FTM-145
Beltramo 3
Peptide 234
2
b
2
c
2
2
a
d-Pya(4) refers to d-4-pyridinylalanine
b
Gly = Leu refers to an alkenyl replacement for the conventional carboxamide;
c
Glyψ[Tz]Leu refers to a triazolyl replacement for the conventional carboxamide. See references for details.
et al. described a number of assays developed for high throughput
screening purposes including the use of an IP3 sensitive IP-One
HTRF™ assay. [11] It is worth noting that each of these assays has
a marked difference in the kinetics of the outputs and so the degree
of exposure of the cells to test compounds also differs.
Here we assessed kisspeptin analogues via an alternate in vitro
reporter assay. We transiently transfected HEK293T cell line with a
human GPR54 construct (hGPR54) and monitored the activity of
GPR54 using the dual luciferase reporter assay, in which the expres-
sion of firefly luciferase gene is activated by the induction of the se-
rum response element (SRE)–mitogen–activated protein (MAP)
kinase signal transduction pathway.
Auspep. N,N-diisopropylethylamine (DIPEA), DMF, DCM, were pur-
chased from Merck. Triisopropylsilane (TIPS) was purchased from
Sigma-Aldrich. 4-Fluorobenzoic acid was purchased from Alfa
Aesar. Rhodamine B isothiocyanate was obtained from Sigma-
Aldrich. The azidopentanoylpiperazine-rhodamine B derivative
was prepared in-house. [26,27] Cy5.5 carboxylic acid was obtained
from Lumiprobe (Cat# 27090) (Florida). All chemicals were used
without further purification.
Liquid Chromatography Mass Spectra (LCMS) were acquired on a
Shimadzu 2020 LCMS system incorporating a photodiode array detec-
tor coupled directly into an electrospray ionisation source and a single
quadrupole mass analyser. RP-HPLC was carried out at room temper-
ature employing a Phenomenex Luna C8 (100 × 2.0 mm I.D.) column
eluting with a gradient of 0–100% ACN in 0.05% aqueous TFA over
15 min at a flow rate of 0.2 ml/min. Mass spectra were obtained in pos-
itive mode with a scan range of 200–2000 m/z. Semi-preparative
reverse-phase HPLC was performed using a Waters Associates liquid
chromatography system (Model 600 controller and Waters 486
Tuneable Absorbance Detector) using a gradient of 0 80% ACN in
0.1% TFA over 20 min or 30 min at a flow rate of 10 ml/min on a
Phenomenex Luna C8 100 Å, 10 μm (250×21.2mm I.D.) or
Phenomenex Luna C8 100 Å, 10 μm (100 × 21.2 mm I.D.) column.
Except where otherwise stated peptide syntheses were per-
formed on Rink amide resin (0.3–0.7 meq/g, 100–200 mesh,
0.1 mmol scale) using conventional Fmoc-based solid phase peptide
synthesis. Fmoc-protected amino acids in threefold molar excess
were coupled using DMF as solvent; sixfold molar excess of
diisopropylethylamine in DMF (70 ml/l) with threefold molar excess
of HCTU as the activating agent for 50 min. Fmoc deprotection was
carried out by treatment with 20% piperidine in DMF for 10 min.
Peptide cleavage off resin was performed using a cocktail containing
TFA/TIPS/DMB (92.5%:2.5%:5%) for 2 h [28]. The cleavage mixture
was filtered, concentrated by a stream of nitrogen, precipitated by
cold diethyl ether and centrifuged. The resulting crude product
was dissolved in water/acetonitrile (1 : 1) and lyophilised overnight.
We have used this assay to evaluate the activity of a range of syn-
thetic analogues of KP10. These include analogues possessing a
range of novel structural motifs: peptides incorporating unusual
amino acids, fluorescent KP10 analogues and cyclic peptides. [19]
Kisspeptins have been shown to be degraded by serum proteases
via the cleavage of the terminal Tyr-Asn and Arg-Phe bonds but
47
50
51
also endpeptidase cleavage after Trp Phe and Gly .[20,21] As
such a particular priority of this approach was to target modifica-
tions that protect the products from proteolytic degradation. We
2
decide to examine the β -homoamino acid class, which might offer
reduced peptidase susceptibility. Introduction of β-amino acid ho-
mologues of DNA-encoded α-amino acids has resulted in an array
of interesting pharmacological and structural outcomes in peptide
2
science, although β -homoamino acids have been less well studied
3
than their β -homoamino acid counterparts. [22–24] We also com-
piled a range of fluorescent ligands, targeting a variety of
fluorophores, linking chemistries and positional substitution. [25]
Such ligands as well as providing useful tools for studying KISS1R
pharmacology contribute to the SAR understanding of kisspeptin-
related peptides.
Materials and Methods
Chemistry
2
β -Homoamino Acid Containing Peptides
α
N -Fmoc-protected amino acids were purchased from Auspep and
ChemImpex. Rink amide resin and O-(1H-6-chlorobenzotriazol-1-
y1)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HCTU)
were purchased from ChemImpex. 6-Chloro-benzotriazole-1-
yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock)
was purchased from Merck. Methylbenzhydrylamine (MBHA) resin,
piperidine and trifluoroacetic acid (TFA) were purchased from
2
Boc-β -homoamino acid synthesis
General procedure for the Knoevenagel condensation
To a solution of methyl cyanoacetate I (1.3eq.) and piperidine
(seven drops) in MeOH (50 ml) was added the aldehyde (1eq.)
J. Pept. Sci. 2016; 22: 406–414
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

CAMERINO ET AL.
and the reaction mixture refluxed for 16 h. The reaction was cooled
to room temperature and the product precipitated out by addition
of H O (50 ml), filtered and washed (H O, 10 ml) to give the product.
J= 6.0 Hz, Ar. H); 3.62 (3H, s, COOMe); 3.37–3.35 (1H, m, 0.5× CH2);
3.28–3.26 (1H, m, CH); 2.94–2.89 (2H, m, 0.5 × CH and 0.5× CH );
2
2
2.80–2.78 (1H, m, 0.5 × CH ); 1.54 (9H, s, Boc); 1.42 (9H, s, Boc)
2
2
2
Purification where necessary by column chromatography.
2
Boc-(±)-β -homotryptophan-methyl ester (IIId). From IId (1.00 g,
Methyl 2-cyano-3-phenylacrylate (IIa) [29,30]. From benzaldehyde
4.40mmol). Brown oil (0.898g, 61%). R
f
0.22 (25% EtOAc in hexane).
1
(
2.20 ml, 24.9 mmol). White solid (3.56g, 96%). R 0.66 (25% EtOAc
3
H-NMR: (CDCl , 300MHz) δ 7.85 (1H, d, J = 9.0Hz, Ar. H); 7.34 (1H, d,
f
1
in hexane). H-NMR: (CDCl , 300 MHz) δ 8.26 (1H, s, CH); 7.99 (2H,
J= 9.0 Hz, Ar. H); 7.18 (1H, t, J = 9.0Hz, Ar. H); 7.11 (1H, t, J = 9.0 Hz, Ar.
H); 7.01 (1H, s, Ar. H); 3.67 (3H, s, COOMe); 3.45–3.29 (2H, m, CH );
3.20–3.10 (1H, m, CH); 3.04–2.92 (2H, m, CH ); 1.43 (9H, s, Boc). ESI-
3
d, J = 9.0 Hz, Ar. H); 7.56–7.47 (3H, m, Ar. H); 3.93 (3H, s, COOMe).
Mp: 85–87 °C, lit [30] 87–89 °C.
2
2
2+
+
MS (+): m/z = 333.5 [M + H] , 665.8 [M+ 2H] .
Methyl 2-cyano-4-methylpent-2-enoate (IIb). From isobutyraldehyde
1
(
1.50 ml, 16.4 mmol) Clear oil (0.905 g, 36%). R 0.84 (CHCl ). H-
General procedure for the ester hydrolysis
f
3
NMR: (CDCl , 400 MHz) δ 7.43 (1H, d, J = 10.6 Hz, CH); 3.82 (3H, s,
COOMe); 2.99–2.90 (1H, m, CH); 1.11 (6H, d, J = 6.6 Hz, 2 × CH3).
3
To a solution of the Boc protected methyl-ester (1eq.) in THF (5ml)
was added a solution of lithium hydroxide (1.2eq.) in 5 ml of H O.
2
Methyl 3-(4-hydroxyphenyl)-2-isocyanoacrylate (IIc) [31]. From p-
The reaction mixture was stirred at room temperature for 16 h or
heated with stirring under microwave irradiation (100 °C,
Power= 105 W) for 40 min. The organic solvent was removed in
vacuo, the residue acidified with 1 M HCl to pH2 and extracted with
ethyl acetate (3× 10 ml). The combined organic layers were washed
with brine (10 ml), dried over Na SO and the solvent removed in
hydroxybenzaldehyde (2.40g, 19.7mmol). White solid (3.25 g,
1
8
1%). H-NMR: (CDCl , 300 MHz) δ 8.21 (1H, s, CH); 7.96 (2H, d,
3
J = 8.7Hz, Ar. H); 6.91 (2H, d, J= 9.0Hz, Ar. H); 3.87 (3H, s, COOMe).
Mp: 213–214 °C, lit [31] 208–210 °C.
2
4
Methyl 3-(1H-indol-3-yl)-2-isocyanoacrylate (IId) [32,33]. From 3-
vacuo to give the product.
indolecarboxaldehye (3.60 g, 24.9 mmol). White solid (3.35 g, 75%).
1
2
R
f
0.61 (50% EtOAc in hexane). H-NMR: (CDCl
3
, 300 MHz) δ 9.15
Boc-(±)-β -homophenylalanine (IVa)[36–39]. From IIIa (0.636 mg,
1
(1H, br s. NH); 8.64 (2H, m, Ar. H and CH); 7.87–7.84 (1H, m, Ar. H);
2.17mmol) Yellow solid (0.623 g, > 99%). H-NMR: (CD
300 MHz) δ 7.34–7.20 (5H, m, Ar. H); 3.46–3.20 (2H, m, CH ); 3.14–
2.71 (3H, m, CH and CH
3
OD,
7
.50–7.48 (1H, m, Ar. H); 7.34 (2H, m, Ar. H); 3.93 (1H, s, COOMe).
2
+
À
ESI–MS (+): m/z = 227.1 [M+ H] , ESI-MS (À) 225.1 [MÀH] . Mp:
2
À
); 1.46 (9H, s, Boc). ESI-MS (À): m/z = 278.5
À
186.5–188.5 °C, lit [34] 165.9 °C.
[M-H] , 557.7 (2 MÀH) . Mp: 94À96°C.
2
Boc-(±)-β -homoleucine (IVb)[37]. From IIIb (0.321 mg, 1.24 mmol)
General procedure for one-pot reduction and Boc protection
1
Pale yellow oil (0.301 g, 99%). H-NMR: (CD
3
OD, 300 MHz) δ 3.47–
); 2.77–2.61 (1H, m, CH); 1.91–1.69 (2H, m, CH );
.47 (9H, s, Boc); 0.94 (6H, d, J= 6.48 Hz, 2 × CH
). ESI-MS (À): m/
To a solution of the alkene (1eq.) in MeOH (300 ml) in a 500 ml
round bottomed flask fitted with a drying tube, was added
3
.13 (2H, m, CH
2
2
1
3
À
CoCl
2
2 2
· 6H O (0.5eq.) and Boc O (3eq.). The mixture was cooled to
z = 244.2 [M-H] .
0
°C and NaBH
4
(14 eq.) added slowly over 2 h. The reaction was
2
Boc-(±)-β -homotyrosine (IVc). From IIIc (319 mg, 1.03 mmol). Yel-
warmed to room temperature and stirred for 16–24 h. To the reac-
tion mixture was added diethylenetriamine (1eq.) and stirred for
1
low oil (0.186 g, 61%). H-NMR: (CD
3
OD, 600 MHz) δ 7.00 (2H, d,
J= 6.8 Hz, Ar. H); 6.71 (2H, d, J = 6.9 Hz, Ar. H); 3.43–3.33 (1H, m,
3
0 min. The solvent was evaporated in vacuo and the purple residue
taken up in EtOAc. The organic layer was washed with saturated aq.
NaHCO , and dried over Na SO . The solvent was removed in vacuo
2 2
0.5 × CH ); 3.24–3.22 (1H, m, 0.5 × CH ); 3.13–3.08 (1H, m, CH);
2
.77–2.75 (1H, m, 0.5 × CH
2
); 2.66–2.63 (1H, m, 0.5× CH
2
); 1.43 (9H,
3
2
4
+
À
s, Boc). ESI-MS (+): 318.3 (M+ Na) , ESI-MS (À): m/z = 294.9 [M = H] .
Boc-(±)-β -homotryptophan (IVd) [40]. From IIId (0.856 mg,
and the residue purified by flash chromatography using 0–25%
EtOAc in hexane to give product.
2
1
2
2.57mmol). Yellow oil (0.679 g, 83%). H-NMR: (CDCl
, 300MHz) δ
3
Boc-(±)-β -homophenylalanine-methyl ester (IIIa) [35]. From IIa
7.55 (1H, d, J = 7.7Hz, Ar. H); 7.08 (3H, dt, J = 7.0, 14.6 Hz, Ar. H);
(
0.500 g, 0.27 mmol). Pale yellow oil (0.185 g, 26%). R
f
0.74 (25%
1
6.95 (1H, s, Ar. H); 3.46–3.22 (2H, m, CH ); 3.14 (1H, dd, J = 4.9,
2
EtOAc in hexane). H-NMR: (CDCl
3
, 300MHz) δ 7.28–7.23 (5H, m,
1
3.2Hz, CH); 3.03–2.82 (2H, m, CH ); 1.41 (9H, s, Boc). ESI–MS (À):
2
Ar. H); 4.94 (1H, s, NH); 3.62 (3H, s, COOMe); 3.37–3.20 (2H, m, CH
and CH); 2.97–2.75 (3H, m, CH and CH); 1.41 (9H, s, Boc).
2
-
m/z = 317.2 [M-H] .
2
2
Boc-(±)-β -homoleucine-methyl ester (IIIb). From IIb (0.679 g,
2
β -Homoamino Acid Containing Peptide Synthesis
1
4
.43 mmol). Pale yellow oil (0.321 g, 28%). H-NMR: (CDCl
00 MHz) δ 4.89 (1H, br. s, NH), 3.66 (3H, s, COOMe), 3.29 (1H, dd,
), 3.25–2.59 (3H, m, CH and CH), 1.39 (1H, s,
), 1.29–1.20 (6H, m, 2 × CH ); 0.87 (9H, d, J =6.1 Hz, Boc).
3
,
2
β -Homoamino acid containing peptides were prepared on MBHA
3
resin by a mixed Boc/Fmoc-based synthesis strategy, such that after
J = 11.8, 5.6 Hz, CH
.5 × CH
2
2
2
coupling with Boc-protected β -homoaminoacids, deprotection
0
2
3
+
+
was performed with 100% trifluoroacetic acid. The TFA-stable
Fmoc-Arg(Mtr) was used for introduction of arginine. Peptides were
cleaved from the resin using a mixture of trifluoromethane sulfonic
acid and TFA as previously described.[19]
ESI-MS (+): m/z = 260.4 [M+ H] , 282.4 (M+ Na) .
2
Boc-(±)-β -homotyrosine-methyl ester (IIIc). From IIc (1.72 g,
8
.48 mmol). Pale yellow oil (0.548 g, 16%). R
f
0.54 (50% EtOAc in
, 600 MHz) δ 6.99 (2H, d, J = 8.1Hz, Ar. H);
.71 (2H, d, J= 7.5Hz, Ar. H); 3.66 (3H, s, COOMe); 3.35 (1H, m,
1
hexane). H-NMR: (CDCl
3
6
0
0
Peptide Labelling
.5 × CH
.5 × CH
2
); 3.23 (1H, m, CH); 2.90–2.88 (2H, m, 0.5 × CH
2
and
2
); 2.72 (1H, dd, J = 9.9, 15.6 Hz, 0.5× CH ); 1.43 (9H, s, Boc).
2
Rhodamine linked amide 12 was prepared by treatment of
kisspeptin with a rhodamine B derivative activated with PyClocK
and NMM in DMF for three hours. The solvent was removed in
vacuo, the residue dissolved in a minimum volume of TFA,
2
Di-Boc-(±)-β -homotyrosine-methyl ester was also obtained pale
yellow oil (0.319g, 9%). R
CDCl , 600MHz) δ 7.15 (2H, d, J = 12.0Hz, Ar. H); 7.06 (2H, d,
1
f
0.79 (50% EtOAc in hexane). H-NMR
(
3
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Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2016; 22: 406–414

KISSPEPTIN ANALOGUES
2 2 2 4 2
Scheme 1. Reagents and conditions; i, R-CHO, piperidine, MeOH, reflux 16 h; ii, CoCl .6H O, Boc O, NaBH , 24 h; iii, LiOH, THF/H O, RT, 16 h or mw, 100 nM,
40 min.
precipitated with ether and centrifuged to yield the crude product
which was purified by RP-HPLC. RhB-KP10 12 RT 14.94 min
Cy5.5 linked amide 13 was prepared by treatment of kisspeptin
with Cy5.5 carboxylic acid activated with PyClocK and NMM in
DMF for three hours. The solvent was removed in vacuo, the residue
dissolved in a minimum volume of TFA, precipitated with ether and
centrifuged to yield the crude product which was purified by RP-
HPLC. Cy5.5-KP10 13 RT 12.8 min
Rhodamine thioureas 14 and 16 were prepared by treating a so-
lution of the peptide (0.015mmol) in methanol with rhodamine B
2 3
isothiocyanate (8mg, 0.15 mmol) and 0.1 M Na CO was added un-
til a pH of 9 was attained. The reaction was stirred overnight at RT,
then diluted with water (40 ml) and freeze dried. The residue was
purified by RP-HPLC.
at 100%. Data analysis including the calculation of half effective
concentration (EC ) and a fit sigmoidal graph was performed using
Origin 6.0 software (Microcal Software, Inc., Northampton, MA). All
data are presented as mean ± SEM.
50
Protease Inhibitor Assay
To examine the effect of protein degradation during the assay, dual
luciferase reporter gene assay was conducted with the addition of
Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO). Different
doses (0, 1, 2, and 5 μl) of the protease inhibitor was applied to
500 μl media and HEK293-T cells transfected with the vectors were
incubated with different doses of KP-10 in the media for 6 h. The
cells were harvested and the luciferase activity in the cell extracts
was determined using Dual-Luciferase Reporter Assay System
1
1
4 was obtained as two regioisomers RT 14.7, 15.1 min.
6 was obtained as two regioisomers RT 14.5, 14.9 min.
(Promega) in a single-tube luminometer (Sirius) according to the
manufacturer’s instruction.
The triazolo-linked rhodamine B peptide 15 was prepared by in-
volved dissolving the corresponding peptide-alkyne (1eq.) in H
and adding a solution of the azido substituted rhodamine B deriv-
ative (4eq.) in DMF to give a 1 : 3 ratio of H O to DMF. Copper sul-
2
O
Results
2
fate (10eq.), TBTA (10 eq.) and sodium ascorbate (10eq.) were
then added and the reaction mixed for 3 h. [27] Peptides were pu-
rified by reverse-phase preparative HPLC.
(
a) Synthetic Analogues of KP-10
A variety of kisspeptin analogues were prepared as assay con-
trols. This included a kisspeptin 10 (1), the d-Tyr analogue (2), a sim-
plified analogue of TAK448 (3), an analogue of FTM-080, (4) and
Peptide 234 (5). (Table 1) These peptides were synthesised using
standard Fmoc solid phase peptide synthesis procedures on Rink
amide resin.
Dual Luciferase Reporter Gene Assay
Full CDS region of human GPR54 mRNA (GenBank accession num-
ber NM_032551) was amplified from FirstChoice PCR-Ready Human
Brain cDNA (Ambion, Austin, TX) and cloned into a pcDNA3.1(+) ex-
pression vector (Invitrogen, Carlsbad, CA) to prepare the GPR54 ex-
pression construct (pcGPR54). HEK293-T cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Alckland,
NZ) supplemented with 10% fetal bovine serum (FBS), 0.1 x
penicillin–streptomycin solution (iDNA, Kuala Lumpur, Malaysia)
2
In addition, we evaluated some novel peptides containing β -
homoamino acids. The Boc-protected amino acids were prepared
according to the general methods described by Pataj[41] and
Caddick.[42] The first step of this synthesis (Scheme 1) was the
Knoevenagel condensation between methylcyanoacetate (I), and
the aldehydes in the presence of piperidine and methanol, to give
the alkenes in 36–95% yield. One pot reduction and N-Boc protec-
tion of alkenes IIa-d were performed using cobalt-boride, which
was formed in situ from sodium borohydride and cobalt chloride
hexahydrate. Base-catalysed hydrolysis of the methyl esters IIIa-d
under thermal or microwave heating (100 °C, 105 W) with lithium
under 5% CO . One day before transfection, cells were plated in
2
24-well plates in the media without penicillin–streptomycin.
Cotransfection of pcGPR54 (100 ng/well), pSRE-Luc (100 ng/well;
Stratagene, La Jolla, CA), and pRL-TK vectors (25ng/well; Promega,
Madison, WI) was carried out with Lipofectamine 2000 transfection
reagent (Invitrogen) overnight according to the manufacturer’s in-
structions. The cells were serum starved in the media with 0.5%
FBS for 18–20 h, and then treated with the vehicle or GPR54 ana-
logues in the media for 6 h. The cells were harvested and the lucif-
erase activity in the cell extracts was determined using Dual–
Luciferase Reporter Assay System (Promega) in a single-tube
luminometer (Sirius; Berthold Detection Systems GmbH, Pforzhein,
Germany) according to the manufacturer’s instruction.
2
2
hydroxide gave the Boc-β -homoamino acids, Boc-β -hTyr,
2
2
2
Boc-β -hPhe, Boc-β -hLeu, and Boc-β -hTrp (IVa-d) in good yields.
All spectroscopic data were consistent with proposed structures
and literature values, where available.
2
A KP10 analogue containing a β -homotyrosine at the N-
terminus was prepared as well as analogues of the linear
hexapeptides FTM 180 and 4. The β-amino acids were coupled as
Boc-protected derivatives. In examples that contained mid-
sequence substitutions, were synthesised using a mixed Fmoc
and Boc protection strategy on MBHA resin. The yields of purified
peptide in these cases were quite low, in part due to poor yield of
Data Analysis
2
Luciferase induction as a percentage of maximal compound activity
was calculated by setting the highest induction of each compound
the β -amino acid coupling, but in addition, residual
trifluoromethanesulfonic acid created problems in isolation of the
J. Pept. Sci. 2016; 22: 406–414
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CAMERINO ET AL.
Figure 1. Chemical structure of fluorophores utilised in this study.
product from crude cleavage reaction mixtures. The products were
all obtained as pairs of diastereomers, consistent with the racemic
nature of the precursor β -homoamino acids and the mixtures
could not be separated by HPLC. In the case of peptide 3, the puri-
fication from other impurities resulted in partial resolution of the di-
astereomers yielding a 1 : 4 mixture of the diastereomeric pair.
We also prepared fluorescently labelled analogues where the
Reported compounds 2 and 4 were also found to be potent ag-
onists, giving EC50 values in the low nanomolar range and consis-
tent with literature reports (Peptide 2 EC50 3.6 nM (receptor
binding)[13]; Peptide 4 EC50 3.1 nM Flipr assay[12]). Compound 3,
a simplified analogue of TAK448[14] has not been previously re-
ported and was shown to be a particularly potent agonist, with
an EC50 of 0.24 nM in the reporter assay. (Figure 2B–D)
2
fluorophore was appended either through the amino terminus
It should be noted that one feature of the assays with these tran-
siently transfected cells was that the degree of induction varied
from experiment to experiment, although the EC50 stayed relatively
constant. However, it was also noticed that the level of induction for
analogues 2–3 was greater than that for kisspeptin itself in the
same batch of treatment. We hypothesised that this was not due
to any difference in intrinsic efficacy but due to improved stability
of these compounds compared with kisspeptin in the assay condi-
tions and this was investigated further as described later. As such
the reported EC50 values for a peptide are referenced against the
maximum induction for that same peptide. (Table 1, Figure 2)
The assay system should also be suitable to evaluate potential
antagonism of GPR54, with incubation of test compounds in the
presence of established agonists such as KP-10. The reported
antagonist ‘Peptide 234’ (Ac-d-Ala-Asn-Trp-Asn-Gly-Phe-Gly-d-Trp-
4
(
(
12-14) or by introduction of an appending ligand to replace Asn
15-16). (Figure 1) While this residue is important for activity as
shown by alanine replacement, we reasoned that the carboxamide
side chain might be mimicked by thiourea or triazole functions,
utilising linkage methods we have described in other work.[27]
The fluorescent rhodamine B thiourea derivatives 14 and 16 were
prepared by coupling with either rhodamine B isothiocyanate ei-
ther in solution or on resin. Two regioisomeric products were ob-
tained corresponding to the two regioisomers of the parent
rhodamine isothiocyanate. The N-terminal rhodamine 12 and
Cy5.5 carboxamides 13 were prepared by reacting with the corre-
sponding PyClock-activated esters. The triazolyl linked rhodamine
was prepared by coupling the propargyloxyproline derivative with
an azido functionalised rhodamine using the copper-assisted
alkyne-azide cycloaddition reaction.[27]
2
Arg-Phe-NH )[15] was tested in the assay but showed no antago-
Biological assays
nism of the KP-10 response. This was both with in-house
synthesised samples and the samples obtained from the laboratory
that described the peptide (R. Millar, The Queens Medical Research
Institute, Edinburgh).[15] These samples did display phenotypic be-
haviours indicative of reported GPR54 antagonism in mouse
models (data not shown). The reason for these conflicting results
is unknown – the problem may be due to a technical aspect of
the luciferase assay system or it may relate to an off-target effect
of Peptide 234 that results in indirect but functional antagonism
of Kisspeptin responses.
(a) Powerful Luciferase induction by kisspeptin of KISS1R
transfected HEK293T cells
The specificity of the reporter gene assay system was confirmed
by comparing hGPR54 construct (hGPR54 in a pGEM-T Easy vector)
with a control plasmid. Treatment with human KP-10 (1) increased
relative (Firefly/Renilla) luciferase activity in a dose–response man-
ner (Figure 2A), while no induction was seen in the HEK293T cells
without hGPR54 expression, indicating that the assay system exclu-
sively detects the activation of exogenous GPR54 and is thus useful
in the evaluation of kisspeptin analogues in the present study. The
induction of luciferase activity was time dependent and shown to
be maximal at 6 h of incubation. The EC50 of KP-10 was determined
to be 13 ±1.9 nM, in general accord with the results obtained under
other assay formats.[12,13]
In the study of the modified kisppetin analogues it was observed
2
that both β -homoamino acid and fluorescent labelling could have
dramatic effects on KISS1R agonist activity.
The kisspeptin 10 analogue bearing an N-terminal β -hTyr substi-
2
tution 6 was a potent KISS1R agonist with an EC50 of 0.41 nM.
(Figure 2E) This shows the tolerance the receptor has for the N-
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J. Pept. Sci. 2016; 22: 406–414

KISSPEPTIN ANALOGUES
Figure 2. Dose response curves for luciferase induction by test peptides upon GPR-54 transfected HEK293-T cells. See Materials and Methods for details.
terminal modifications consistent with the potent activity of
analogues 2 and 3.
the peptide has been amenable to certain structural changes – such
as FTM145, which has a non-peptide replacement for the Phe-Gly
linkage and the triazolo linked peptide Beltramo 3 (Table 1
).[16,43] Note also that a series of previously published [19] cyclic
peptides that adopt stable helical structures also showed no ago-
nist efficacy in these assays (data not shown). (Figure 3)
Finally, the fluorescently labelled ligands showed a quite marked
and surprising structure-activity profile. The inclusion of the rhoda-
mine label at the N-terminus of KP-10, as amide 12 or thiourea 14
had little effect upon the observed potency with EC50 values of 30
On the other hand, the β-amino acid containing analogues of
FTM80 and 3 were much less active but agonist activity was seen
at high concentrations of certain ligands. The compounds showed
some differences in EC50 that must correspond to the ability to
adopt the bioactive conformation found with the parent peptide.
2
Compound 8, where a glycine was replaced by a β-alanine (β -
hGly), showed the strongest activity in this series with an EC50 of
0.6 μM. This is consistent with other studies where this region of
J. Pept. Sci. 2016; 22: 406–414
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CAMERINO ET AL.
amino acids might be more resistant to proteases in particular
and so express agonist efficacy throughout the incubation period.
To test this hypothesis, the assay of KP-10 activity was repeated
but in this case with increasing amounts of a protease inhibitor
cocktail present in the culture media. The level of induction in-
creased by over 150% at both 1 nM and 100 nM concentrations sug-
gesting that protease activity was diminishing the full expression of
agonist activity in the reporter assay.
Discussion
The development of new ligands for KISS1R is being pursued by a
number of groups, as the potential for therapeutic agonists and an-
tagonists is evident from numerous studies. In our pursuit of such
ligands we have developed a functional assay format that is robust
and straightforward, giving a clear dose dependent readout of ag-
onist activity. Three reported agonists gave EC50 values consistent
with those reported by others. The activity of peptide 2 has not
been described, and showed it to be a potent agonist of GPR54. No-
tably, this increased potency was achieved by the introduction of D-
Figure 3. Comparison of luciferase induction of GPR-54 transfected
HEK293-T cells by vehicle (DMEM), KP-10 (100 nM) and KP10 (1 nM) in the
presence of 0, 1, 2 or 5 μl of protease inhibitor cocktail.
and 10 nM, respectively. The inclusion of the Cy5.5 label 11
completely abolished activity. This form of the Cy5.5 label has a
non-sulfated benzo[e]indol-2-ylidene rendering it quite hydropho-
bic, which may impact on the binding affinity. (Table 2)
Introduction of a rhodamine label replacing the asparagine resi-
due of the TAK-488 analogues resulted in a potent inhibitor 16
2
amino acid, d-Tyr or the β-amino acid, β hTyr at the N-terminus
with relatively little change to the C-terminal sequence, although
it may reflect either increased binding affinity or be due to an in-
creased stability in the time course assay. This is consistent with
1
(EC50 = 1 nM) albeit less potent than the parent molecule. The intro-
the reported improvement of in vivo potency of d-Tyr -KP-10.[13]
duction of a triazolyl-proline at the same position in dTyr-KP10 15
abolished activity, a likely consequence of the conformational ef-
fect of the proline-like residue as the precursor peptide was simi-
larly inactive (data not shown).
The variation in the extent of luciferase induction by the variety
of peptide agonists described earlier was further examined. While
there might be a number of mechanisms to explain ‘partial’
agonism displayed by KP-10, one hypothesis was that the peptide
was being degraded over the course of the incubation period, such
that the actual concentration of agonist was diminishing across
time. As such the synthetic analogues that possessed modified
We have also shown that agonist potency can be retained with
the inclusion of fluorophores, also providing other potentially use-
ful tools for studying the kisspeptin/GPR54 system. Kaneda et al. re-
cently described alternate fluorescently labelled kisspeptin
analogues through N-terminal derivatization of KP-14 and KP-52.
The retention of agonist activity with the tetramethylrhodmaine
and rhodamine green labels is consistent with our data here.[25]
We also examined the use of β-amino acids as a means of
constraining the short pentapeptide agonists in the hope that it
may confer metabolic stability while also retaining a bioactive con-
former. The changes to the structure proved very deleterious to
Table 2. LC-MS and EC50 data for KISS1R peptides
Number
(KP-10)
Sequence
ESI-MS m/z
1302.8
EC50 /(nM)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
H-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
H-d-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
Ac-d-Tyr-Hyp-Asn-Thr-Phe-Gly-Leu-Arg-Trp-NH
Amb-Phe-Gly-Leu-Arg-Phe- NH
Ac-d-Ala-Asn-Trp-Asn-Gly-Phe-Gly-d-Trp-Arg-Phe-NH
2
13 ± 1.9
5.3 ± 1.23
0.24 ± 0.012
16 ± 5.6
a
2
652.2
2
1210.4
771.6
1295.6
1300.8
774.6
774.7
786.0
774.3
812.9
2
2
—
2
H-β hTyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
2
0.41 ± 0.065
Inactive
2
FBz-β hPhe-Gly-Leu-Arg-Phe- NH
FBz-Phe-βAla-Leu-Arg-Phe- NH
2
2
600 ± 140
2
b
Amb-Phe-Gly-β hLeu-Arg-Phe- NH
2
>1000
2
0
FBz-Phe-Gly-Leu-Arg-β hPhe- NH
2
Inactive
2
b
1
2
3
4
5
6
FBz-Phe-Gly-Leu-Arg-β hTrp- NH
2
>1000
a
RhB-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
Cy5.5-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
RhB(NHCS)-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH
2
948.9
31 ± 19.2
Inactive
a
2
934.7
a
2
902.10
10 ± 11.0
Inactive
c
2
a
H-d-Tyr-Asn-Trp-Rtp-Ser-Phe-Gly-Leu-Arg-Phe-NH
Ac-d-Tyr-Hyp-Dap(γSCNH-RhB)-Thr-Phe-Gly-Leu-Arg-Trp-NH
989.0
a
2
842.2
1.0 ± 0.49
a
2+
ESI-MS m/z = (M + 2H)
;
b
Luciferase induction still rising at maximum concentration (10 μm) tested;
Rtp = rhodamine-triazolylproline (Figure 1) [27]
c
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J. Pept. Sci. 2016; 22: 406–414

KISSPEPTIN ANALOGUES
9
Orsini MJ, Klein MA, Beavers MP, Connolly PJ, Middleton SA, Mayo KH.
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alogues irrespective of the EC50 of the compound. This an
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The authors would like to thank the Director General of Health
Malaysia for his permission to publish this article and the Director
of the Institute for Medical Research for his support. This study
was supported by Ministry of Health Malaysia, JPP07-034 (to I.S.P.).
The provision of Australian Post-graduate Awards (to M.A.C and
M.L.) is acknowledged.
2
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